Apparatus and methods for multi-step channel emulsification

ABSTRACT

Methods and devices for forming droplets are provided. In certain embodiments, the methods and devices form droplets having different diameters.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/133,621 filed Mar. 16, 2015 and 62/269,289 filed Dec. 18,2015, the contents of each of which are incorporated herein byreference.

FIELD OF THE INVENTION

Embodiments of the present invention relate to methods and devices forforming droplets.

BACKGROUND

The following descriptions and examples are not admitted to be prior artby virtue of their inclusion within this section.

Compartmentalization is a technique that is becoming increasinglypopular in the molecular diagnostics and life science research fields.Applications include digital polymerase chain reaction (PCR), two-stagePCR multiplexing (including genotyping), single-cell analysis, targetedsequencing, multiplex immunoassays, ultra-sensitive immunoassays, andlibrary prep for sequencing. Each separate application places differentdemands on the number of compartments, monodispersity of eachcompartment, and the volume of each compartment.

One approach for compartmentalizing reactions is by using droplets,which are isolated volumes of a first fluid that are completelysurrounded by a second fluid or by a second fluid and one or moresurfaces. In the molecular diagnostics and life science research fieldsthis is typically two immiscible liquids. Techniques for dropletgeneration include co-flow, flow focusing, and T-junction. Co-flowdroplet generation forms droplets via pinching of the inner flow from anorifice in a co-flow design as described by, for example, David Weitz(“Monodisperse emulsion generation via drop break off in a coflowingstream,” Langmuir, 2000). Stone and Weitz (“Monodisperse doubleemulsions generated from a microcapillary device,” Science, 2005)demonstrated double emulsions using a modified co-flowing technique.Flow focusing uses a co-flow design which is geometrically confined inthe channel to produce droplets (see, e.g., Stone, “Formation ofdispersions using “flow focusing” in microchannels,” APL, 2003).T-junction droplet generation methods and modifications thereof (e.g.,Y-junction, cross junction, ψ-junction) generally involve intersectingflows of continuous and dispersed phases (see, e.g., Quake, “Dynamicpattern formation in a vesicle-generating microfluidic device”, PRL,2001; and Weitz, D. A., Stone, H., “Geometrically mediated breakup ofdrops in microfluidic devices,” PRL, 2004). Additionally, U.S. Pat. No.7,943,671 (incorporated herein by reference) described a stepemulsification technique that employed an abrupt change in the aspectratio of a single microchannel to rapidly destabilize a confinedco-flowing stream.

The droplet generation techniques described above all require flows ofboth continuous and dispersed phases. In contrast, Sugiura et al.described a technique in which droplet formation was driven largely byinterfacial tension (Sugiura, S., Nakajima, M. “Interfacial tensiondriven monodispersed droplet formation from microfabricated channelarray,” Langmuir, 17:5562-5566 (2001)). With this technique, dropletsare generated via falling off a ledge after ejection from a fluidicchannel. More recently, Dangla et al., have also described techniquesfor generating droplets by modulating the interfacial curvature betweenimmiscible liquids using a sloped ceiling to produce a continuouslyincreasing gap height, called a gradient of confinement (U.S. Pat. Pub.2013/0078164 (incorporated herein by reference); Dangla et al., “Dropletmicrofluidics driven by gradients of confinement,” PNAS, 10(3):853-858(2013)). This gradient of confinement has similarities with theinterfacial curvature modulation achieved with a discrete step asdescribed by Sugiura et al. (see above).

SUMMARY OF THE INVENTION

Exemplary embodiments of the present disclosure relate to systems andmethods for forming droplets, including a multi-step microchannelemulsification device.

One embodiment provides an emulsification device comprising: a channelhaving an inlet portion; a first step in fluid communication with theinlet portion; a second step in fluid communication with the first step;and a third step in fluid communication with the second step. In someembodiments, the emulsification device comprises a plurality of inletportions, a single continuous first step or a plurality of first stepsthat are each in fluid communication with an inlet portion in theplurality of inlet portions; a single continuous second step or aplurality of second steps that are each in fluid communication witheither the single continuous first step or a first step in the pluralityof first steps, and a single continuous third step or a plurality ofthird steps that are each in fluid communication with the single secondstep or a second step in the plurality of second steps.

The channel having the inlet portion has a channel height CH and a widthCW. In certain embodiments, CW is greater than CH, while in otherembodiments CH is greater than CW. In particular embodiments, the ratioof CW/CH is between 0.1 to 10.0, 0.2 to 8.0, 0.5 to 5.0, 1.0 to 4.0, 2.0to 4.0, or 2.5 to 3.5. In certain embodiments, the ratio of CW/CH isabout 3.0.

Specific embodiments include a first step in fluid communication withthe inlet portion, where the first step has a tread length T1 and a stepheight SH1. In certain embodiments, SH1 is greater than CH by a riserheight R1, where R1 is greater than zero. In specific embodiments, theratio of SH1/CH is greater than 1.0 and less than 10.0, or moreparticularly greater than 1.0 and less than 5.0, or more particularlygreater than 1.0 and less than 4.0, or more particularly still greaterthan 1.0 and less than 2.0. In particular embodiments, the ratio ofSH1/CH is approximately 1.5. Specific embodiments include a second stepin fluid communication with the first step, where the second step has atread length T2 and a step height SH2. In particular embodiments, SH2 isgreater than SH1 by a riser height R2, where R2 is greater than zero. Incertain embodiments, the ratio of SH2/CH is greater than 1.0 and lessthan 10.0, or more particularly greater than 1.0 and less than 5.0, ormore particularly greater than 1.0 and less than 3.0. In particularembodiments, the ratio of SH2/CH is approximately 2.0. Particularembodiments include a third step in fluid communication with the secondstep, where the third step has a step height SH3 that is greater thanSH2 by a riser height R3, where R3 is greater than zero. In certainembodiments, the ratio of SH3/CH is greater than 1.0 and less than 15.0,or more particularly greater than 1.0 and less than 10.0, or moreparticularly greater than 5.0 and less than 10.0. In particularembodiments, the ratio of SH3/CH is approximately 7.5.

In particular embodiments, R1 is greater than 0.1 micron and less than1000 microns, greater than 1.0 micron and less than 100 microns, greaterthan 5.0 microns and less than 100 microns, greater than 5.0 microns andless than 50 microns, greater than 1.0 micron and less than 50 microns,greater than 1.0 micron and less than 20 microns, greater than 3.0microns and less than 30 microns, or greater than 5.0 microns and lessthan 20.0 microns. In certain embodiments, R1 is at least 5.0 microns.In some embodiments, R1 is about 5, 10, or 20 microns, or any rangederivable therein. In particular embodiments, R2 is greater than 0.1micron and less than 1000 microns, greater than 1.0 micron and less than100 microns, greater than 5.0 microns and less than 100 microns, greaterthan 5.0 microns and less than 50 microns, greater than 1.0 micron andless than 50 microns, greater than 1.0 micron and less than 20 microns,greater than 3.0 microns and less than 30 microns, or greater than 5.0microns and less than 20.0 microns. In certain embodiments, R2 is atleast 5.0 microns. In some embodiments, R2 is about 5, 10, or 20microns, or any range derivable therein. In some embodiments, R1 isequal to R2. In particular embodiments, R3 is greater than 0.1 micronand less than 1000 microns, greater than 1.0 micron and less than 1000microns, greater than 5.0 microns and less than 1000 microns, greaterthan 5.0 microns and less than 500 microns, greater than 10.0 micronsand less than 1000 microns, greater than 10.0 micron and less than 500microns, greater than 50 microns and less than 300 microns, or greaterthan 100.0 microns and less than 1000.0 microns. In some embodiments, R3is about 55, 110, or 275 microns, or any range derivable therein. Incertain embodiments, R3 is at least 55.0 microns. In certainembodiments, R3 is at least 275 microns. In particular embodimentsconfigured to produce different size droplets, CH will be 10 microns, 20microns, and 50 microns, and R1 will equal R2 and will be 5 microns, 10microns, and 25 microns. In certain embodiments configured to producedifferent size droplets, R3 will be 55 microns, 110 microns and greaterthan 275 microns. In some embodiments, R1 is greater than R2, and R2 isgreater than R3. In other embodiments, R3 is greater than, R2, and R2 isgreater than R1. In some embodiments, R1=R2=R3. In yet otherembodiments, R1=R2, and R3 is greater than R1. In some embodiments, theratio of R3/R1 is at least 10.0. In some embodiments, the ratio of R3/R2is at least 10.0.

In specific embodiments, the ratio of T1/CH is between 0.1 and 7, ormore particularly greater 1 and less than 5, or more particularlygreater than 3.0 and less than 4.0. In certain embodiments, the ratio ofT1/CH is greater than 1.0. In specific embodiments, the ratio of T2/CHis between 0.1 and 7, or more particularly greater 1 and less than 5, ormore particularly greater than 3.0 and less than 4.0. In certainembodiments, the ratio of T2/CH is greater than 1.0. In certainembodiments the ratio of T2/CH is less than T1/CH.

In certain embodiments of the emulsification device, CH is between 1micron and 50 microns, or more particularly between 5 microns and 30microns, or more particularly between 6 and 20 microns, or moreparticularly between 8 and 12 microns, or still more particularlyapproximately 10 microns. In certain embodiments, CH is at least 5microns, 10 microns, 20 microns, or 50 microns.

In particular embodiments, the first step has a width W1 greater thanCW, the second step has a width W2 greater than CW, and the third stephas a width W3 that is greater than CW. In certain embodiments, thefirst step has a width W1 greater than CW, the second step has a widthW2 equal to W1, and the third step has a width W3 that is greater thanW1. In certain embodiments, W1=W2=W3. Particular embodiments include aplurality of inlet portions, where each inlet portion in the firstplurality of inlet portions has a height CH and a width CW. In certainembodiments, the ratio of CW/CH is greater 1.0 for each inlet portion.In certain embodiments, the ratio of CW/CH is between 0.1 to 10.0, 0.2to 8.0, 0.5 to 5.0, 1.0 to 4.0, 2.0 to 4.0, or 2.5 to 3.5 for each inletportion. In certain embodiments, the ratio of CW/CH is about 3.0 foreach inlet portion. Certain embodiments include a plurality of firststeps, wherein each first step in the plurality of first steps is influid communication with an inlet portion in the plurality of inletportions, and has a length T1 and a height SH1, where SH1 is greaterthan CH by a riser height R1. Some embodiments include a continuousfirst step in fluid communication with the inlet portions in theplurality of inlet portions, and have a length T1 and a height SH1,where SH1 is greater than CH by a riser height R1. In particularembodiments, the ratio of SH1/CH is greater than 1.0. In specificembodiments, the ratio of SH1/CH is greater than 1.0 and less than 10.0,or more particularly greater than 1.0 and less than 5.0, or moreparticularly greater than 1.0 and less than 4.0, or more particularlystill greater than 1.0 and less than 2.0. In particular embodiments, theratio of SH1/CH is approximately 1.5. Particular embodiments include aplurality of second steps, where each second step in the plurality ofsecond steps is in fluid communication with a first step in theplurality of first steps or a single continuous first step, is in fluidcommunication with the third step, and has a length T2 and a height SH2,where SH2 is greater than SH1 by a riser height R2. Some embodimentsinclude a single continuous second step in fluid communication with thesingle continuous first step or the plurality of first steps, is influid communication with the third step, and has a length T2 and aheight SH2, where SH2 is greater than SH1 by a riser height R2. Inparticular embodiments, the ratio of SH2/CH is greater than 1.0. Incertain embodiments, the ratio of SH2/CH is greater than 1.0 and lessthan 10.0, or more particularly greater than 1.0 and less than 5.0, ormore particularly greater than 1.0 and less than 3.0. In particularembodiments, the ratio of SH2/CH is approximately 2.0. In someembodiments, the single continuous second step or plurality of secondsteps is in fluid communication with a common third step. Otherembodiments include a plurality of third steps, where each third step isin fluid communication with a single continuous second step or a secondstep of a plurality of second steps.

In some embodiments, the emulsification device has a single inletportion. In other embodiments, the emulsification device has a pluralityof inlet portions. In specific embodiments, the emulsification devicehas 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,150, 200, 250, 500, or 1000 inlet portions, or any range derivabletherein. The plurality of inlet portion may each be in fluidcommunication with one or more of a dedicated first, second, and/orthird step in a plurality of first, second, and/or third steps or theplurality of inlet portions may be in fluid communication with one ormore of a common first, second, and/or third step.

In some embodiments, the emulsification device has a single nozzle. Inother embodiments, the emulsification device has a plurality of nozzles.In specific embodiments, the emulsification device has 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, or1000 nozzles, or any range derivable therein. The plurality of nozzlesmay each be in fluid communication with a dedicated third step in aplurality of third steps or the plurality of nozzles may be in fluidcommunication with a common third step. Two or more nozzles of theplurality of nozzles may have geometries configured to form droplets oftwo or more different sizes. For example, an emulsion device may havethree populations of nozzles or channels in which a first population hasthe geometries CH=10 microns, R1=5 microns, R2=5 microns, a secondpopulation has the geometries CH=20 microns, R1=10 microns, R2=10microns, and a third population has the geometries CH=50 microns, R1=25microns, R2=25 microns, whereby the first, second, and third populationsof nozzles produce droplets of about 45 microns, 120 microns, and 300microns, respectively. The various sized droplets may be collected in acommon third step region or the various sized droplets may be collectedin separate third step regions with other droplets of the same size.Where the device contains a plurality of spatially separated third stepregions, each third step may have a riser height R3 and or step heightSH3 that is different from one or more of the other third step regions.By way of illustration, in the example discussed above with droplets ofabout 45 microns, 120 microns, and 300 microns, the value of R3 forthree distinct third step regions could be 55 microns, 110 microns, and275 microns.

Certain embodiments include a method of forming an emulsion using anemulsification device according to the present disclosure. In particularembodiments of the method, the first step, the second step and the thirdstep of the device contain a first fluid that is substantially static.Specific embodiments of the method include introducing a second fluidinto the inlet portion and through the first step, the second step andthe third step. In particular embodiments, a partial droplet of thesecond fluid forms in the first step, a complete droplet of the secondfluid forms in the second step (or during the transition between theplurality of first steps and the second steps), and the complete dropletof the second fluid is directed from the second step to the third step.

In some embodiments, the complete droplet of the second fluid iscompressed in the second step such that a height of the complete dropletin the second step is less than a length of the complete droplet in thesecond step. In specific embodiments, the complete droplet of the secondfluid is compressed in the third step such that a height of the completedroplet in the third step is less than a length of the complete dropletin the third step. In certain embodiments in which the complete dropletof the second fluid is compressed in the third step, the dropletdiameter (at its shortest dimension or “height”)<SH3<2× the dropletdiameter (at its longest dimension or “length”). In some embodiments thecomplete droplet of the second fluid is not compressed in the third stepsuch that a height of the complete droplet in the third step is equal tothe length of the complete droplet in the third step. In particularembodiments, the height of the complete droplet in the second step isless than height of the complete droplet in the third step. Inparticular embodiments, the length of the droplet forming on the firststep is greater than T1. In particular embodiments, the length of thedroplet on the droplet forming on the second step is greater than T2. Incertain embodiments, the second fluid contains an analyte of interest.In specific embodiments, the second fluid contains one or more assayreagents, and in particular embodiments, the assay reagent is apolymerase chain reaction (PCR) primer, a salt, or an enzyme. In certainembodiments, the length of a droplet on step one and step two is morethan the respective tread lengths (e.g. T1 and T2) such that a portionof the droplet that is in contact with the step surface will also be incontact with the step edge on that step.

In some embodiments, the first fluid is an oil. In specific embodiments,the first fluid is a hydrophobic liquid and the second fluid is ahydrophilic liquid. In other embodiments, the first fluid is ahydrophilic liquid and the second fluid is a hydrophobic liquid. Inparticular embodiments, either the first fluid or the second fluidcomprises an emulsifying agent, and in certain embodiments, theemulsifying agent comprises a non-ionic surfactant and/or a blockingprotein.

In some embodiments of the method, a complete droplet of the secondfluid forms in the second step at a rate of at least 10 completedroplets per second. In some embodiments of the method, a completedroplet of the second fluid forms in the second step at a rate ofbetween 1 and 30 complete droplets per second, or more particularly at arate of between 10 and 30 complete drops per second, or moreparticularly at a rate of approximately 12, 13, 14, 15, 16, 17, 18, 19,or 20 droplets per second. In some embodiments, a plurality of nozzlesare employed to produce at least 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 1500, 2000, 3000, 4000, 5000, or any range derivable therein,droplets per emulsion device per second.

In certain embodiments, the complete droplet of second fluid has anaverage diameter between 40 and 400 microns, 45 and 300 microns, or 40and 50 microns. Certain embodiments are configured to produce dropletshaving different diameters, including for example droplets withdiameters of 20-60, 80-160, and 200-400 microns. Particular embodimentsare configured to produce droplets having different diameters, includingfor example droplets with diameters of 45, 120, and 300 microns. Inspecific embodiments, the emulsion formed between the first fluid andthe second fluid has a monodispersity (the deviation of the dropletdiameter) of less than ten percent. In particular embodiments, theemulsion formed between the first fluid and the second fluid has amonodispersity of one, two, three, four, five, six, seven, or eightpercent, or any range derivable therein.

In certain embodiments, the channels and/or steps can be etched insilicon. In particular embodiments, the etched silicon can be coveredwith glass and/or plastic polymer (plastic, elastomer, rubber,polycarbonate, cyclo-olefin Polymer [COP], etc.), e.g.polydimethylsiloxane (PDMS). In some embodiments, the surfaces of thechannel and/or steps may be coated with a hydrophobic composition. Inspecific embodiments, the hydrophobic composition isperfluorodecyltrichlorosilane (FDTS).

Certain embodiments include a method of forming an emulsion, the methodcomprising obtaining an emulsification device comprising a firstplurality of channels each having an inlet portion, a first step, asecond step, and a third step; a second plurality of channels eachhaving an inlet portion, a first step, a second step, and a third step;and a third plurality of channels each having an inlet portion, a firststep, a second step, and a third step, wherein the plurality of firststeps, the plurality of second steps and the plurality of third stepsfor the first, second and third pluralities of channels contain a firstfluid that is substantially static. Exemplary embodiments of the methodmay further comprise: introducing a second fluid into the plurality ofinlet portions and through the plurality of first steps, the pluralityof second steps and the plurality of third steps of the first, secondand third pluralities of channels, where: a partial droplet of thesecond fluid forms in each of the plurality of first steps of the first,second and third pluralities of channels; a first complete droplet ofthe second fluid forms in a transaction between the plurality of firststeps and the plurality of second steps in each of the first pluralityof channels; a second complete droplet of the second fluid forms in atransaction between the plurality of first steps and the plurality ofsecond steps in each of the second plurality of channels; and a thirdcomplete droplet of the second fluid forms in a transaction between theplurality of first steps and the plurality of second steps in each ofthe third plurality of channels; the second complete droplet of thesecond fluid is larger than the first complete droplet of the secondfluid; and the third complete droplet of the second fluid is larger thanthe second complete droplet of the second fluid.

In particular embodiments of the method: the diameter of the firstcomplete droplet is between 25 μm and 65 μm; and the diameter of thesecond complete droplet is between 80 μm and 200 μm; and the diameter ofthe third complete droplet is between 200 μm and 400 μm. In specificembodiments of the method: the diameter of the first complete droplet isbetween 35 μm and 55 μm; the diameter of the second complete droplet isbetween 100 μm and 140 μm; and the diameter of the third completedroplet is between 250 μm and 350 μm. In certain embodiments of themethod, the diameter of the first complete droplet is approximately 45μm; and the diameter of the second complete droplet is approximately 120μm; and the diameter of the third complete droplet is approximately 300μm.

The term “coupled” is defined as connected, although not necessarilydirectly, and not necessarily mechanically. Two items are “coupleable”if they can be coupled to each other, and, when coupled, may still becharacterized as “coupleable.” Unless the context explicitly requiresotherwise, items that are coupleable are also decoupleable, andvice-versa. One non-limiting way in which a first structure iscoupleable to a second structure is for the first structure to beconfigured to be coupled (or configured to be coupleable) to the secondstructure.

The terms “a” and “an” are defined as one or more unless this disclosureexplicitly requires otherwise.

The term “substantially” and its variations (e.g., “approximately” and“about”) are defined as being largely but not necessarily wholly what isspecified (and include wholly what is specified) as understood by one ofordinary skill in the art. In any disclosed embodiment, the terms“substantially,” “approximately,” and “about” may be substituted with“within [a percentage] of” what is specified, where the percentageincludes 0.1, 1, 5, and 10 percent.

The terms “comprise” (and any form of comprise, such as “comprises” and“comprising”), “have” (and any form of have, such as “has” and“having”), “include” (and any form of include, such as “includes” and“including”) and “contain” (and any form of contain, such as “contains”and “containing”) are open-ended linking verbs. As a result, a method ordevice that “comprises,” “has,” “includes” or “contains” one or moresteps or elements possesses those one or more steps or elements, but isnot limited to possessing only those one or more elements. Likewise, astep of a method or an element of a device that “comprises,” “has,”“includes” or “contains” one or more features possesses those one ormore features, but is not limited to possessing only those one or morefeatures. For example, a system that comprises four channels may havemore than four channels.

A “fluid” generally refers to a substance that tends to flow and toconform to the shape of its container. The fluid may have any suitableviscosity that permits flow. Where two or more fluids are present in avolume, the fluids may be, for example, miscible, slightly miscible, orimmiscible. As used herein, two fluids are immiscible, or not miscible,with each other when one is not soluble in the other under theconditions at which the emulsion is used.

As used herein, a “droplet” is an isolated volume of a first fluid thatis completely surrounded by a second fluid or is completely surroundedby a second fluid and one or more surfaces. Non-limiting examples ofdroplets include a hydrophilic liquid suspended in a hydrophobic liquid,a hydrophobic liquid suspended in a hydrophilic liquid, and a gas bubblesuspended in a liquid.

An “emulsion” is a suspension of a liquid in a liquid. In someembodiments, the emulsion may be a “microemulsion” or a “nanoemulsion,”i.e., an emulsion in which the dispersed phase has an average diameteron the order of micrometers or nanometers, respectively. An emulsion maybe created, for example, by allowing droplets of the desired size orsizes to enter into a solution that is immiscible with the droplets. Incertain embodiments, a fluidic stream or fluidic droplets may beproduced on the microscale in a microchannel (i.e., a channel or stephaving an average cross-sectional dimension of between about 1 μm to 1mm).

A fluid that is “substantially static” is a fluid in which flow-inducedpressure variations are negligible. For example, in various embodimentsdisclosed herein a first fluid is substantially static in a channel anda second fluid, which is immiscible with the first fluid, flows into thechannel via an inlet. The second fluid may be caused to flow through theinlet by, for example, a pump. The substantially static first fluid mayhave some movement due to displacement of the first fluid by the secondfluid flowing into the channel; but there is no additional inletconveying a flow of the first fluid into the channel. There may,however, be an outlet or waste channel to accommodate any of the firstfluid that is displaced from the channel by the second fluid. In otherwords, the first fluid is “passive.” Also, because the first fluid ispassive and does not co-flow with the second fluid, the flow rate doesnot determine droplet size as it does in other co-flow droplet formationtechnologies such as T-junction devices.

The inlet portion, first step, and second step may be referred tocollectively herein as a “nozzle.” An emulsification device may have asingle nozzle or a plurality of nozzles. A plurality of nozzles may bein fluid communication with a common third step or a plurality ofnozzles may each be in fluid communication with a plurality of separatedthird steps. A plurality of nozzles will have a plurality of inletportions, but the first step may be a single continuous step in fluidcommunication with the plurality of inlet portions or the first step maybe a plurality of structurally distinct first steps each in fluidcommunication with a dedicated inlet portion of the plurality of inletportions. Likewise, the second step may be a single continuous step influid communication with the first step or first steps, or the secondstep may be a plurality of structurally distinct second steps each influid communication with a dedicated first step of a plurality of firststeps.

Furthermore, a device or structure that is configured in a certain wayis configured in at least that way, but may also be configured in waysthat are not listed. Metric units may be derived from the English unitsprovided by applying a conversion and rounding to the nearestmicrometer.

The feature or features of one embodiment may be applied to otherembodiments, even though not described or illustrated, unless expresslyprohibited by this disclosure or the nature of the embodiments.

Any embodiment of any of the disclosed devices and methods can consistof or consist essentially of—rather thancomprise/include/contain/have—any of the described elements and/orfeatures and/or steps. Thus, in any of the claims, the term “consistingof” or “consisting essentially of” can be substituted for any of theopen-ended linking verbs recited above, in order to change the scope ofa given claim from what it would otherwise be using the open-endedlinking verb.

Other features and associated advantages will become apparent withreference to the following detailed description of specific embodimentsin connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings illustrate by way of example and not limitation.For the sake of brevity and clarity, every feature of a given structuremay not be labeled in every figure in which that structure appears.Identical reference numbers do not necessarily indicate an identicalstructure. Rather, the same reference number may be used to indicate asimilar feature or a feature with similar functionality, as maynon-identical reference numbers.

FIG. 1A is a perspective view of an exemplary embodiment of a multi-stepemulsification device according to the present disclosure.

FIG. 1B is a perspective view of an exemplary embodiment of a multi-stepemulsification device according to the present disclosure.

FIG. 2 is a section view of the embodiment of FIG. 1B.

FIGS. 3A-3C are section views of the embodiment of FIG. 1B duringoperation.

FIG. 4 is a perspective view of a device comprising a plurality ofnozzles according to the present disclosure.

FIG. 5 is a graph illustrating dispersion percentage versus flow ratefor a network of single-step emulsification devices and a network ofmulti-step emulsification devices.

FIG. 6 is a graph illustrating dispersion percentage versus flow rate ascalculated for a single-step emulsification device and a multi-stepemulsification device of the networks of devices from FIG. 5.

FIG. 7 is a chart illustrating droplet size dispersion percentage for asingle-step emulsification device and a multi-step emulsification device

FIG. 8 is a perspective view of an exemplary embodiment of a multi-stepemulsification device according to the present disclosure.

FIG. 9 is a partial section view of the embodiment of FIG. 8.

FIG. 10 is a partial section view of the embodiment of FIG. 8.

FIG. 11 is a partial section view of the embodiment of FIG. 8.

FIG. 12 is a schematic of droplets formed by the exemplary embodiment ofFIG. 8.

FIG. 13 is a schematic showing different dynamic ranges for varyingdroplet sizes and areas.

FIG. 14 is a perspective view of an exemplary embodiment of a multi-stepemulsification device according to the present disclosure.

DETAILED DESCRIPTION

Various features and advantageous details are explained more fully withreference to the non-limiting embodiments that are illustrated in theaccompanying drawings and detailed in the following description. Itshould be understood, however, that the detailed description and thespecific examples, while indicating embodiments of the invention, aregiven by way of illustration only, and not by way of limitation. Varioussubstitutions, modifications, additions, and/or rearrangements willbecome apparent to those of ordinary skill in the art from thisdisclosure.

In the following description, numerous specific details are provided toprovide a thorough understanding of the disclosed embodiments. One ofordinary skill in the relevant art will recognize, however, that theinvention may be practiced without one or more of the specific details,or with other methods, components, materials, and so forth. In otherinstances, well-known structures, materials, or operations are not shownor described in detail to avoid obscuring aspects of the invention. Itis understood that for purposes of clarity, not all reference numbersare shown for every component visible in each figure.

It should be understood that the present devices and methods are notintended to be limited to the particular forms disclosed. Rather, theyare to cover all modifications, equivalents, and alternatives fallingwithin the scope of the claims.

FIG. 1A illustrates an emulsification device 190 in perspective view. Inthis embodiment, emulsification device 190 comprises an inlet channel195 with a channel width CW and a channel height CH. Inlet channel 195further comprises portions of increasing width. For example, inletchannel 195 comprises a portion with a width W1 that is greater thanwidth CW. In particular embodiments, the ratio of W1/CW may be greaterthan 2.0, greater than 5.0, greater than 10.0, greater than 50.0, orgreater than 100.0. In specific embodiments the ratio of W1/CW may bebetween 5.0 and 25.0. In the embodiment shown, inlet channel 195comprises further portions with increasing widths W2 and W3.

FIGS. 1B and 2 respectively illustrate an emulsification device 100 inperspective and section views. The embodiment in FIG. 1B and FIG. 2includes a channel 105 with risers 110, 120 and 130 and steps 101, 102and 103 as described further below. For purposes of clarity, not allelements are labeled in both FIG. 1B and FIG. 2. In the embodiment inFIG. 1B, channel 105 includes an inlet portion having a channel width CWand a channel height CH. Channel 105 further includes a portion having awidth W1 that is greater than CH. In some embodiments, a plurality ofinlet portions may be in fluid communication with a common first step,in which case the width W1 of this common step would be significantlygreater than the width CW of any individual inlet portion. The width W1of this common step would be greater than the sum of all widths of theinlet portions in fluid communication with the step FIGS. 3A-3Cillustrate section views of emulsification device 100 during operation.

In the embodiment shown in FIG. 1B and FIG. 2, emulsification device 100comprises a multi-step configuration comprising a channel 105 having aninlet portion 107, a first step 101, a second step 102, and a third step103, each in fluid communication with the other. In addition,emulsification device 100 comprises a first riser 110 at the interfaceof inlet portion 107 and first step 101, a second riser 120 at theinterface of first step 101 and second step 102, and a third riser 130at the interface of second step 102 and third step 103. First step 101comprises a first step height SH1 and a first tread length T1, secondstep 102 comprises a second step height SH2 and a second tread lengthT2, and third step 103 comprises a third step height SH3 and a thirdtread length T3.

As used herein, the tread length T1 equals the distance between firstriser 110 and second riser 120, tread length T2 equals the distancebetween second riser 120 and third riser 130, and tread length T3 equalsthe distance between third riser 130 and the end of a droplet collectionchamber of the emulsification device 100 that is distal from inletportion 107 or tread length T3 equals the distance between third riser130 and a fourth riser if the emulsion device comprises one or moreadditional steps. In addition, SH1 equals the distance between opposingsurfaces 115 and 111, SH2 equals the distance between opposing surfaces115 and 112, and SH3 equals the distance between opposing surfaces 115and 113. In the illustrated embodiment, surface 115 is distal fromfirst, second and third risers 110, 120 and 130. In the embodimentshown, surface 111 extends between first and second risers 110 and 120,and surface 111 is parallel to surface 115. Similarly, surface 112extends between second and third risers 120 and 130, and surface 112 isparallel to surface 115 in this embodiment. Furthermore, surface 113 isparallel to surface 115 and extends from riser 130 to the end ofemulsification device 100 that is distal from inlet portion 107. In theillustrated embodiment, first, second and third risers 110, 120 and 130are perpendicular to surface 115.

As explained further below, the dimensions and geometry of the channeland steps are configured to produce highly monodispersed emulsions athigh frequency from a single fluid flow. As demonstrated by the datapresented below, a multi-step configuration can provide significantimprovement in monodispersity over a single-step design. Without wishingto be bound by theory, it is believed that the inferior performance ofthe single-step design can be attributed to the fact that the formingdroplets would contact previously formed droplets in unpredictable ways,thus affecting the forming droplet size. The multi-step configurationdisclosed herein solves the inferior monodispersity issue of thesingle-step design. In particular, the multi-step configuration definesmultiple sections serving specific functions in droplet formation.

In the illustrated embodiment, inlet portion 107 comprises a channelheight CH and a channel width CW (shown in FIG. 1). In particularembodiments, the ratio of CW/CH is greater than 0.2 and less than 5.0.Inlet portion 107 is in fluid communication with first step 101, whichcomprises a tread length T1 and a step height SH1, where SH1 is greaterthan channel height CH by a riser height R1 that is greater than zero.In specific embodiments, the ratio of first step height to channelheight SH1/CH is greater than 1.0 and less than 5.0. While exemplaryembodiments shown and described herein include nozzles arranged in alinear configuration, other embodiments may include different nozzlearrangements, including for example, a circular arrangement of nozzles.

It is understood that dimensional terms, such as height, width, andlength are used for reference purposes only and not intended to requirea particular orientation of microchannel emulsification device 100. Asused in reference to FIGS. 2 and 3A-3C, height refers to a verticaldimension (e.g. top to bottom of the illustration page), width refers toa dimension perpendicular to the plane of the illustrated section view(e.g. perpendicular to the page), and length refers to the a horizontaldimension (e.g. left to right of the page). In general, the terms heightand length refer to perpendicular dimensions in one plane, while theterm width refers to a dimension perpendicular to the plane of theheight and length.

In the embodiment shown, SH1 is greater than CH by riser height R1 (e.g.the dimensional difference between SH1 and CH). In addition, SH2 isgreater than SH1 by a riser height R2, and SH3 is greater than SH2 by ariser height R3. The various riser heights R1, R2 and R3 are shownextending downward in the vertical direction in FIG. 2. It isunderstood, however, that the riser heights may also extend in theupward or side direction. The droplet remains confined (i.e.,non-spherical) in the nozzle and, therefore, surface tension, notgravity, is the primary force affecting droplet formation duringoperation of exemplary embodiments, allowing the riser heights to beformed in any direction (e.g., downward, upward, or side) if desired.Either CH or CW (whichever dimension is smaller) are primary factors indetermining the diameter of droplets formed by device 100, and SH1 or W1are secondary factors in determining the diameter of droplets formed bydevice 100.

In the illustrated embodiment, inlet portion 107 of channel 105 has awidth-to-height ratio (CW/CH) that is greater than 0.2 and less than5.0. In certain embodiments, the ratio of CW/CH may be greater than 1.5and less than 4.5, and in particular embodiments, the ratio of CW/CH maybe greater than 2.5 and less than 3.5, and in specific embodiments theratio of CW/CH may be approximately 3.0.

As previously mentioned, first step 101 is in fluid communication withinlet portion 107, and height SH1 of first step 101 is greater than CHby a riser height R1. In exemplary embodiments, the ratio of SH1/CH isgreater than 1.0 and less than 5.0, or greater than 1.25 and less than2.75 or greater than 1.5 and less than 2.5, or greater than 1.75 andless than 2.25, or greater than 1.25 and less than 1.75.

Emulsification device 100 also comprises a second step 102 in fluidcommunication with first step 101, and height SH2 of second step 102 isgreater than SH1 by a riser height R2. In exemplary embodiments, theratio of SH2/CH is greater than 0.1 and less than 5.0. In addition,emulsification device 100 also comprises a third step 103 in fluidcommunication with second step 102, and step height SH3 of third step103 is greater than SH2 by a riser height R3. In exemplary embodiments,R3 is greater than 0.1 micron and less than 1000 microns. In theembodiment shown, R1 is greater than R2, and R2 is greater than R3.However, in other embodiments R3 is greater than R2, and R2 is greaterin R1. In some embodiments, R3 is greater than R2, and R2 is equal toR1. Furthermore, the embodiment shown includes a ratio of T1/CH between0.1 and 7.0 and a ratio of T2/CH that is less than T1/CH.

In particular embodiments, the ratio of T1/R1 is greater than 2.0, orgreater than 5.0 or greater than 10.0. In certain embodiments the ratioof T2/R2 is greater than 2.0, or greater than 5.0 or greater than 10.0.

FIGS. 3A-3C illustrate emulsification device 100 during operation. Forpurposes of clarity, not all elements of emulsification device 100 arelabeled in FIGS. 3A-3C. Reference can be made to FIG. 2 for elements notlabeled in FIGS. 3A-3C. FIG. 3A illustrates a partial droplet 152transitioning from first step 101 to second step 102. FIG. 3Billustrates a droplet 153 transitioning from second step 102 to thirdstep 103. FIG. 3C illustrates a droplet 154 on third step 103. Referringinitially to FIG. 3A, during operation a fluid stream 151 can beintroduced (e.g., directed in a flowing stream from a higher pressure toa lower pressure) into inlet portion 107. Inlet portion 107 isconfigured to deliver a sample thread 151 to first riser 110 with heightR1 between inlet portion 107 and first step 101.

In particular embodiments, fluid stream 151 may be a sample threadcomprising a hydrophilic liquid, while steps 101, 102 and 103 contain afluid 155 that is a hydrophobic liquid. In some embodiments, fluidstream 151 may comprise a hydrophobic liquid, while fluid 155 comprisesa hydrophilic liquid. In certain embodiments, steps 101, 102 and 103 arefilled with fluid 155 comprising a hydrophobic liquid (e.g. an oil)prior to the introduction of fluid stream 151 comprising a hydrophilicliquid (e.g. an aqueous fluid) into first channel 101. In exemplaryembodiments, fluid 155 is substantially static when fluid stream 151 isintroduced into inlet portion 107 of first channel 105. Further examplesof the types of liquids that may be used for droplet formation inemulsification device 100 are provided below in the section entitled“EMULSIONS”.

First step 101 is configured to begin destabilization of a fluid steam151 (e.g., transitioning a contiguous fluid stream 151 to contain adiscontinuity), and partial droplet 152 is formed in first step 101. Incertain embodiments, partial droplet 152 is approximately ninety percentformed (as measured by volume) in first step 101 during operation. Inexemplary embodiments, first step 101 does not provide for completedroplet formation, and partial droplet 152 is fluidicly connected tofluid stream 151. As shown in FIG. 3A, partial droplet 152 is fluidiclyconnected to fluid stream 151 by a region 158 that has a smallercross-sectional area than fluid stream 151 or partial droplet 152 (e.g.,fluid stream 151 necks down into region 158 before forming partialdroplet 152). Partial droplet 152 is compressed by first step 101 andextends the entire height SH1 of first step 101. According to fluiddynamics and physics principles, partial droplet 152 will seek thelowest possible energy state (e.g. an uncompressed state). Accordingly,partial droplet 152 will continue to progress toward second step 102until it is contact with first riser 110, where the droplet will be lesscompressed than it is in first step 101 due to second step 102 having aheight SH2 that is greater than first step 101 height SH1.

In exemplary embodiments, partial droplet 152 will form a completedroplet 153 (as shown in FIG. 3B) upon reaching second riser 120 at theinterface of first step 101 and second step 102. During operation, fluidstream 151 will be introduced into first channel 101 over a period oftime. When one partial droplet 152 progresses to form a complete droplet153, a subsequent partial droplet will form in first step 101. Secondstep 102 and riser 120 (with riser height R2) are configured to form acomplete droplet 153 that is separated from a partial droplet 152 (andfluid stream 151).

Second step 102 is also configured to provide a protection zone betweenfirst step 101 where partial droplets 152 are forming and third step 103where complete droplets 154 are stored. As such, contact between formed,complete droplets and forming, partial droplets is reduced oreliminated. The lengths of steps 101 and 102 (e.g. dimensions T1 and T2)are sized to accommodate the droplet in those respective sections sothat each section can properly accomplish its function. Theoreticalcalculations for desired droplet formation and advancement indicate atread length T1=3.807(CH) and a tread length T2=1.8585(CH). Actual treadlengths may vary from the dimension theoretically calculated. This is incontrast to a continuous ramp configuration or a configuration with aseries of steps arranged to approximate a ramp where the function ofeach step is identical. Moreover, the multi-step channel disclosedherein also provides manufacturing options that are not available for acontinuous ramp configuration by allowing for increased tolerances.

In exemplary embodiments, complete droplet 153 is compressed within step102 such that it is not completely spherical in shape. For example,complete droplet 153 has a height DH3 equivalent to SH2 (the height ofstep 102, shown in FIG. 2). In addition, droplet 153 also has a lengthDL3 that is greater than DH3. According to fluid dynamics and physicsprinciples, droplet 153 will seek the lowest possible energy state (e.g.an uncompressed state). Accordingly, droplet 153 will continue toprogress toward third step 103 until it reaches second riser 120, wherethe droplet will be less compressed than it is in second step 102 due tothird step 103 having a height SH3 that is greater than second step 102height SH2.

In the embodiment shown, third step 103 is configured to provide storageand, optionally, imaging of droplet 154 via an imaging device 157 (e.g.a camera or photosensitive detector). Additional information regardingoptional imaging of droplet 154 is provided below in the sectionentitled “DROPLET IMAGING.” Droplet 154 is also a complete droplet thatmay or may not be completely spherical, but is less compressed thandroplet 153. Accordingly, droplet 154 height DH4 is greater than droplet153 height DH3, but usually less than droplet 154 length DL4. It isunderstood that FIG. 3C is a section view and multiple droplets 154 canbe located in third step 103 during operation.

FIG. 4 illustrates an exploded assembly perspective view of anemulsification device 250 that comprises a plurality of nozzles 200. Inexemplary embodiments, each nozzle 200 may comprise a channel and stepswith features equivalent to those described herein for the channel andsteps of emulsification device 100. In the embodiment shown,emulsification device 250 comprises nine parallel nozzles 200. Otherembodiments may comprise a greater or fewer number of nozzles. In theembodiment shown, emulsification device 250 comprises a base 220comprised of polydimethylsiloxane (PDMS) and a cover 210 comprised ofglass.

FIG. 5 graphically illustrates dispersion percentage versus flow ratefor a network of sixteen single-step emulsification devices and anetwork of sixteen multi-step nozzles. The single-step emulsificationdevices comprised a step height of approximately 189 μm. CellProfilersoftware and an imaging processing pipeline were used to detectfluorescently labeled droplets. Multiple images were acquired duringeach test, with approximately 300 droplets each. The software createdfiles with a list of all droplets found along with associated dropletdiameters for all files, and the average and standard deviation of thediameters were then calculated. The dispersion was calculated andcompared between the two configurations, where the dispersion is thecoefficient of variation (CV) of the diameter, and where the CV is equalto the standard deviation divided by the mean diameter. The channeldimensions used during this test included a channel height of 20 μm, achannel width of 60 μm, and a ratio of CH/SH1=0.666 or R1/CH=0.5.

FIG. 6 is a graph illustrating dispersion percentage versus flow rate ascalculated for a single-step emulsification device and a multi-stepemulsification device as shown in FIG. 4.

FIG. 7 is a chart illustrating droplet size dispersion percentage for asingle-step emulsification device and a multi-step emulsificationdevice. The channel dimensions used during this test included a channelheight of 25 μm, a channel width of 60 μm, and a delta height/heightequal to 0.5.

In certain embodiments, an emulsification device can be configured togenerate multiple droplet sizes. For example, an emulsification devicemay comprise multiple sets of nozzles and channels with differentgeometries to generate droplets with different sizes and volumes.

Referring now to FIG. 8, an emulsification device 500 comprises a firstplurality of nozzles 300 and a second plurality of nozzles 400. In theembodiment shown, nozzles 300 are supplied fluid with a fluid supplychannel 355, while nozzles 400 are supplied fluid with a fluid supplychannel 455. Droplets formed by nozzles 300 and 400 are collected incollection chambers 350 and 450, respectively. Device 500 furthercomprises a waste channel 550 configured to allow waste material (e.g.excess fluid or droplets) to exit emulsification device 500 and bedirected to a waste collection chamber. In exemplary embodiments, eachnozzle 300 and 400 may comprise a channel and steps with featuresequivalent to those of other embodiments described herein. For example,each nozzle in first and second plurality of nozzles 300 and 400 maycomprise a channel 305 and 405, as shown in FIGS. 9 and 10,respectively. Channels 305 and 405 can be configured with featuresequivalent to those described herein for the channels and steps ofemulsification device 100.

During operation, nozzles 300 and 400 can be configured to generatedroplets having different diameters. For example, each channel 405 canbe configured to generate droplets with a diameter that is greater thanthe diameter of droplets generated from each channel 305. In addition,emulsification device 500 can be configured to control the number ofdroplets generated by each plurality of channels 305 and 405. Forexample, fluid supply channels 355 and 455 can be configured such tocontrol the amount of fluid supplied to channels 305 and 405. In certainembodiments, fluid supply channels 355 and 455 may have differentdiameters, lengths, and/or other factors that can affect the resistanceof fluid flow through the channels and bias the amount of fluid flow tochannels 305 and 405. In other embodiments, fluid supply channels 355and 455 may comprise valves that can be manipulated to control theamount of fluid flow to channels 305 and 405. Such configurations canprovide differing amounts of fluid flow to channels 305 and 405,allowing for different numbers of droplets to be formed by channels 305and 405. The ability to individually control the fluid flow to channels305 and 405 can be used to precisely control the percentage of smallerdiameter droplets formed by channels 405 and the percentage of largerdiameter droplets formed by channels 305. The ability to generatedroplets of different sizes can provide significant advantages overother emulsification devices that generate droplets of generallyequivalent sizes. For example, the different size droplets generated byemulsification device 500 can be used to increase the dynamic rangeavailable during a digital PCR analysis.

In particular embodiments, channels 305 and 405 may have geometriessimilar to those of previously described embodiments. For example asshown in the cross-section view of FIG. 9, channel 305 has an inletportion 307, a first step 301, a second step 302, and a third step 303,each in fluid communication with the other. In addition, channel 305comprises a first riser 310 (with riser height R31) at the interface ofinlet portion 307 and first step 301, a second riser 320 (with riserheight R32) at the interface of first step 301 and second step 302, anda third riser 330 (with riser height R33) at the interface of secondstep 302 and third step 303. First step 301 comprises a first stepheight FSH1 and a first tread length T31, second step 302 comprises asecond step height FSH2 and a second tread length T32, and third step303 comprises a third step height FSH3 and a third tread length T33.

In the embodiment shown in FIG. 9, FSH1 equals the distance betweenopposing surfaces 315 and 311, FSH2 equals the distance between opposingsurfaces 315 and 312, and FSH3 equals the distance between opposingsurfaces 315 and 313. In the illustrated embodiment, surface 315 isdistal from first, second and third risers 310, 320 and 330. In theembodiment shown, surface 311 extends between first and second risers310 and 320, and surface 311 is parallel to surface 315. Similarly,surface 312 extends between second and third risers 320 and 330, andsurface 312 is parallel to surface 315 in this embodiment. Furthermore,surface 313 is parallel to surface 315 and extends from riser 330 to theend of emulsification device 300 that is distal from inlet portion 307.In the illustrated embodiment, first, second and third risers 310, 320and 330 are perpendicular to surface 315.

Referring now to the cross-section view of FIG. 10, channel 405 has aninlet portion 407, a first step 401, a second step 402, and a third step403, each in fluid communication with the other. In addition, channel405 comprises a first riser 410 (with riser height R41) at the interfaceof inlet portion 407 and first step 401, a second riser 420 (with riserheight R42) at the interface of first step 401 and second step 402, anda third riser 430 (with riser height R43) at the interface of secondstep 402 and third step 403. First step 401 comprises a first stepheight SSH1 and a first tread length T41, second step 402 comprises asecond step height SSH2 and a second tread length T42, and third step403 comprises a third step height SSH3 and a third tread length T43.

In the embodiment shown in FIG. 10, SSH1 equals the distance betweenopposing surfaces 415 and 411, SSH2 equals the distance between opposingsurfaces 415 and 412, and SSH3 equals the distance between opposingsurfaces 415 and 413. In the illustrated embodiment, surface 415 isdistal from first, second and third risers 410, 420 and 430. In theembodiment shown, surface 411 extends between first and second risers410 and 420, and surface 411 is parallel to surface 415. Similarly,surface 412 extends between second and third risers 420 and 430, andsurface 412 is parallel to surface 415 in this embodiment. Furthermore,surface 413 is parallel to surface 415 and extends from riser 430 to theend of emulsification device 400 that is distal from inlet portion 407.In the illustrated embodiment, first, second and third risers 410, 420and 430 are perpendicular to surface 315.

In exemplary embodiments, SSH1 (the first step of the “second” channel[e.g. channel 405]) is larger than FSH1 (the first step height of the“first” channel [e.g. channel 305]). In addition, SSH2 (the “second”channel's second step height) is larger than FSH2 (the “first” channel'ssecond step height). Accordingly, the geometry of channels 405 isconfigured to form droplets having a diameter that is larger than thediameter of droplets formed by channels 305. In particular embodiments,SSH1 is at least fifty percent greater than FSH1, and in certainembodiments SSH1 is at least one hundred percent greater than FSH1. Inaddition, SSH2 may be at least fifty percent greater than FSH2 in someembodiments. Such geometries can allow channels 405 to produce dropletshaving a diameter that is at least fifty percent greater than thediameter of droplets produced by channels 305. In certain embodiments,SSH2 may be at least one hundred percent greater than FSH2, allowingchannels 405 to produce droplets having a diameter that is at least onehundred percent greater than the diameter of droplets produced bychannels 305. In particular embodiments, SSH3 may be equal to FSH3 asthe droplets formed by channels 405 and 305 are directed to a commonarea in emulsification device 500 (it is understood the drawings in thefigures are not to scale unless otherwise noted).

In particular embodiments such as those described in FIG. 9, thediameter of droplets formed by channels 305 may be primarily determinedby the dimension of CH or CW of inlet portion 307, whichever is smaller,and secondarily determined by FSH1 as previously described in thediscussion of FIGS. 1-3.

Referring now to FIG. 11, an alternative embodiment for channels 305 isshown. This embodiment is equivalent to the embodiment shown anddescribed in FIG. 9, with the exception that the value of FSH3 is notequivalent to that of SSH3 and therefore comprises an additional step toprovide for advancement of the droplets.

The volume of each droplet formed by channels 305 and 405 isproportional to the cube of the diameter of the droplet (assuming aspherical droplet). In the embodiments shown in FIGS. 9 and 10, if thesmaller of dimensions CH and CW for inlet portion 407 is at least fiftypercent greater than the smaller of dimensions CH and CW for inletportion 307, then the diameter of a droplet formed by channel 405 is atleast fifty percent greater than the diameter of a droplet formed by305. As a result, the volume of a droplet formed by a channel 405 is atleast 3.375 times greater than the volume of a droplet formed by achannel 305. Similarly, if the smaller of dimensions CH and CW for inletportion 407 is at least one-hundred percent greater than the smaller ofdimensions CH and CW for inlet portion 307, then the diameter of adroplet formed by channel 405 is at least twice the diameter of adroplet formed by 305. Consequently, the volume of a droplet formed by achannel 405 is at least eight times greater than the volume of a dropletformed by a channel 305. It is understood that the dimensional ratiosdescribed herein are merely exemplary, and that other embodiments maycomprise channel dimensions with values other than those provided inthis disclosure.

Referring now to FIG. 12, a schematic of droplets formed by channels 405and 305 is shown to include different size droplets. In this embodiment,a plurality of droplets 454 are formed by channels 405, while aplurality of droplets 354 are formed by channels 305. As shown, eachdroplet in the plurality of droplets 454 comprises a diameter D4 that islarger than the diameter D3 of each of the droplets 354. In thisembodiment, D3 is determined by dimension CH or CW of inlet portion 307.Similarly, D4 is determined by dimension CH or CW of inlet portion 407.

The ability to generate droplets of varying volumes such as those shownin FIG. 12 can provide numerous benefits during digital PCR analysis.For example, the use of multiple volume droplets provides for a greaterdynamic range for a given amount of space and overall volume.

In systems using droplets of uniform volume, the upper limit of thedetection is primarily controlled by the volume of each droplet. Thelower limit of the detection is generally controlled by the totalvolume, and therefore number of droplets produced in uniform dropletsystems. Therefore, a large dynamic range in a uniform droplet systemrequires a very large number of small volume droplets. By producingdroplets of varying volumes, the dynamic range can be increased for agiven volume and area provided as compared to uniform droplet systems.The upper limit of detection can be raised by using droplets withdecreased volume. In addition, the lower limit of detection can bereduced by using droplets with increased volume, which allows for theprocessing of greater sample volumes in the same area.

FIG. 13 provides a graphic illustration of the different dynamic rangesfor different areas. As shown in the figure, a low dynamic range (e.g.4-5 logs) of droplets with a 120 μm diameter requires 317 squaremillimeters. A high dynamic range (e.g. 7-8 logs) without dilution orreapportionment with droplets of 27 μm diameter requires 1,413 squaremillimeters. A similarly high dynamic range with 100× dilution in twochambers with droplets of 120 μm diameter requires 634 squaremillimeters. In contrast, a 7-8 logs high dynamic range using twochamber reapportionment with droplets of both 27 μm diameter and 120 μmdiameter only requires 373 square millimeters of space. Accordingly, theuse of droplets with different diameters can provide for higher dynamicranges and/or less space required as compared to systems utilizingdroplets of uniform size and volume.

While previously-described embodiments are configured to producedroplets of one or two different diameters, other embodiments may beconfigured to produce droplets of three or more different diameters.Referring now to FIG. 14, an emulsification device 900 comprises aplurality of channels 600, 700, and 800 with fluid supply channels 655,755 and 855, respectively. In addition, device 900 comprises collectionchambers 650, 750 and 850 for channels 600, 700, and 800, respectively.In exemplary embodiments, collection chambers 650, 750 and 850 may havedifferent heights to accommodate droplets of different diameters. Device900 further comprises a waste channel 950 configured to allow wastematerial (e.g. excess fluid or droplets) to exit emulsification device900 and be directed to a waste collection chamber.

Emulsions

Various embodiments disclosed herein employ a water-in-oil emulsioncomprising a plurality of aqueous droplets in a non-aqueous continuousphase. All or a subset of the aqueous droplets may contain an analyte ofinterest. Emulsions are formed by combining two immiscible phases (e.g.,water and oil), often in the presence of one or more surfactants. Basictypes of emulsions are oil-in-water (o/w), water-in-oil (w/o), andbi-continuous. In droplet-based biological assays, the emulsion willtypically be a water-in-oil emulsion with the assay reagents (e.g., PCRprimers, salts, enzymes, etc.) contained in the aqueous phase. The “oil”phase may be a single oil or a mixture of different oils. Any suitablenon-aqueous fluid may form the non-aqueous continuous phase of theemulsions disclosed herein. In some embodiments, the non-aqueouscontinuous phase comprises a mineral oil, a silicone oil, or afluorinated oil (e.g., Fluorinert® FC-40 [Sigma-Aldrich]).

The emulsion may be stabilized by the inclusion of one or moreemulsifying agents, which act at the water/oil interface to prevent ordelay separation of the phases. Emulsifying agents may also be used toinhibit the merging of adjacent droplets on an array. The compositionsdisclosed herein may also contain one or more emulsifying agent. Inparticular embodiments, the emulsifying agent comprises a non-ionicsurfactant or a blocking protein. Non-limiting examples of non-ionicsurfactants include Tween 20 (polysorbate 20), Triton™ X-100(4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol), Span® 80(sorbitane monooleate), sorbitan monooleate, sorbitan monostearate,polyoxyethylemesorbitan monooleate, and octylphenoxyethoxyethanol. Ionicsurfactants such as sodium cholate, sodium taurocholate, and sodiumdeoxycholate may also be used as emulsifying agents. Additional examplesof emulsifying agents include chemically inert silicone-basedsurfactants such as polysiloxane-polycetyl-polyethylene glycolcopolymer; fluorosurfactants such as perfluorinated polyethers (PFPE)and PFPE-PEG co-polymers; and cholesterol. Non-limiting examples ofblocking proteins include serum albumins, such as bovine serum albuminand acetylated bovine serum albumin.

In certain embodiments, the emulsion is prepared such that variousreagents or analytes are contained within the droplets of the emulsion.In certain embodiments, certain analytes or reagents may be attached toa solid support that also is disposed within the droplet. For example,probes and/or primers may be attached to a solid support. Such solidsupports may be, for example, microspheres (e.g., beads) or otherparticles such as microparticles, gold or other metal nanoparticles,quantum dots, or nanodots. In certain aspects, the particles may bemagnetic, paramagnetic, or super paramagnetic. Examples of microspheres,beads, and particles are illustrated in U.S. Pat. No. 5,736,330 toFulton, U.S. Pat. No. 5,981,180 to Chandler et al., U.S. Pat. No.6,057,107 to Fulton, U.S. Pat. No. 6,268,222 to Chandler et al., U.S.Pat. No. 6,449,562 to Chandler et al., U.S. Pat. No. 6,514,295 toChandler et al., U.S. Pat. No. 6,524,793 to Chandler et al., and U.S.Pat. No. 6,528,165 to Chandler, which are incorporated by referenceherein.

Droplet Imaging

In exemplary embodiments, the droplets may be imaged by a variety oftechniques. To facilitate imaging, the composition containing thedroplets may be dispersed on a surface such that the droplets aredisposed substantially in a monolayer on the surface. The imagingsurface may be, for example, on a slide or in a chamber, such as aglass, plastic, or quartz chamber. The droplets, as well as labeledanalytes or reaction products within the droplets, may be detected usingan imaging system. For example, detecting labeled amplification productsmay comprise imaging fluorescent wavelengths and/or fluorescentintensities emitted from the labeled amplification products. Inembodiments where the droplets contain encoded particles, such asencoded microspheres, the imaging may comprise taking a decoding imageof the encoded particles and taking an assay image to detectamplification products in the droplets. A comparison of the decodingimage and the assay image permits greater multiplex capabilities byusing combinations of fluorophores. The methods of the present inventionmay further comprise correlating the signal from the directly orindirectly labeled amplification product with the concentration of DNAor RNA in a sample. Examples of imaging systems that could be adaptedfor use with the methods and compositions disclosed herein are describedin U.S. Pat. No. 8,296,088 and U.S. Pat. Publ. 2012/0288897, which areincorporated herein by reference.

The droplets may be illuminated with any suitable light source. Thelight source may be configured to provide widespread illumination (i.e.,illumination provided over all or a relatively large area of the imagingregion simultaneously) using light emitted by light sources such aslight emitting diodes (LEDs) or lasers and delivered to the imagingregion directly or via an optical waveguide. Alternatively, theillumination source may be configured to provide illumination of arelatively small spot in the imaging region, and the system may beconfigured to scan the relatively small spot across the imaging region.In this manner, the illumination may be configured as a relatively “tinyflying spot” of focused light generated from one or more LED's, one ormore lasers, one or more other suitable light sources, or somecombination thereof. Imaging the illuminated droplets may comprisedetecting light emitted or reflected from the imaging region of thechamber with a photosensitive detector. Non-limiting examples ofphotosensitive detectors include a photomultiplier tube (PMT), avalanchephoto diode, CCD, CMOS or Quantum Dot camera.

The droplets may comprise labeling agents including, but not limited to,fluorophores, quantum dots, rare earth metals, and chemiluminescentcompounds. The labeling agents may be free floating, attached to ananalyte, attached to a reagent (e.g., a primer, probe, or antibody),attached to a magnetic particle, or any combination thereof. In certainembodiments, the labeling agent is one or more labeled primers or adsDNA-binding dye. In one embodiment, the one or more labeled primerscomprise a fluorophore/quencher pair or a FRET pair.

An imaging chamber may be composed of a single type of material ormultiple materials. In some embodiments, at least a portion of theimaging chamber includes an optically clear material (such as, but notlimited, to optically clear glass, plastic, or quartz), particularly inthe vicinity of the imaging region such that an illumination beam maypass through the imaging chamber to image droplets in the imagingregion. In some cases, a back portion of the imaging chambercorresponding to at least the imaging region may be configured toprovide negligible reflectance and transmittance with respect towavelengths of light emitted by the illumination system.

Assays

Numerous types of assays on a wide range of analytes may be performedinside of droplets. The analyte disposed within a droplet may be anyanalyte of interest including, without limitation, nucleic acids(including DNA or RNA), proteins (including enzymes or antibodies),hormones, carbohydrates, and cells. Depending on the type of analytethat is being detected, amplified, evaluated, etc., additionalcomponents may be disposed in the droplet. For example, where theanalyte is a target nucleic acid, the aqueous droplets may furthercomprise one or more PCR reagents, such as primers, polymerase, MgCl₂,buffer, labeling agent, and/or dNTPs. In one embodiment, one species ofprimer is attached to a solid support disposed within the droplet. Thesolid support may be, for example, a microsphere or nanosphere. As afurther example, where the analyte is a protein the aqueous droplets mayfurther comprise one or more of an antibody, an enzyme, an enzymesubstrate, a labeling agent, and/or BSA. To facilitate detection,analytes or reaction products may be directly or indirectly labeled witha labeling agent such as fluorophores, quantum dots, rare earth metals,and chemilumenescent compounds. The labeling agents may be freefloating, attached to an analyte, attached to a reagent (e.g., a primer,probe, or antibody), attached to a magnetic particle, or any combinationthereof. In certain embodiments, the labeling agent is one or morelabeled primers or a dsDNA-binding dye. In one embodiment, the one ormore labeled primers comprise a fluorophore/quencher pair or a FRETpair. In some embodiments, the labeling agent comprises astreptavidin-conjugated enzyme and a fluorogenic substrate. In oneembodiment, the streptavidin-conjugated enzyme is astreptavidin-conjugated beta-galactosidase and the fluorogenic substrateis a resorufin beta-D-galactopyranoside.

The polymerase chain reaction (PCR) is an example of a reaction that maybe performed within a droplet. In particular, droplets are useful indigital PCR (dPCR) techniques. dPCR involves partitioning the samplesuch that individual nucleic acid molecules contained in the sample arelocalized in many separate regions, such as in individual wells inmicrowell plates, in the dispersed phase of an emulsion, or arrays ofnucleic acid binding surfaces. Each partition (e.g., droplet) willcontain 0 or greater than zero molecules, providing a negative orpositive reaction, respectively. Unlike conventional PCR, dPCR is notdependent on the number of amplification cycles to determine the initialamount of the target nucleic acid in the sample. Accordingly, dPCReliminates the reliance on exponential data to quantify target nucleicacids and provides absolute quantification. Bead emulsion PCR, whichclonally amplifies nucleic acids on beads in an emulsion, is one exampleof a dPCR technique in which the reactions are portioned into droplets.See, e.g., U.S. Pat. Nos. 8,048,627 and 7,842,457, which are herebyincorporated by reference. When dPCR is performed in an emulsion asdiscussed in more detail below, the emulsion should be heat stable toallow it to withstand thermal cycling conditions.

There are various ways of performing dPCR in an emulsion. For example,in one approach a DNA sample is diluted to an appropriate concentration,mixed with PCR reagents (primers, dNTPs, etc.) and encapsulated indroplets in an emulsion as described above, resulting in a number ofdiscrete reaction samples. The droplets are subjected to PCR thermalcycling and the amplicons detected by florescence (or other suitablereporter) imaging as described above.

In another approach, an encoded microsphere is also contained in thedroplet. The microsphere may be used to anchor a primer. By anchoringdifferent primers to different encoded microspheres, each differentprimer, and the corresponding amplicon, may be identified by the encodedmicrosphere to which it is attached. An example of bead emulsion PCR isdescribed in U.S. Pat. No. 8,048,627, which is incorporated herein byreference. It should be noted, however, that the technique described inthe '627 patent involves breaking the emulsions and then isolating beadswith a magnet in order to analyze the sequences on the beads. Incontrast, amplicons may be detected within droplets (e.g., withouthaving to break the emulsion) using the methods and compositiondescribed in the present disclosure.

The thermal cycling of the droplets may be performed by any suitabletechnique known in the art. For example, the droplets may be thermalcycled in a tube or chamber than can be heated and cooled. In someembodiments, the methods employ continuous-flow amplification to amplifythe nucleic acid template. Various methods of continuous flowamplification have been reported. For example, U.S. Pat. No. 7,927,797,which in incorporated herein by reference, describes a water-in-oilemulsion used in conjunction with a continuous flow PCR. Continuous flowof the emulsion across a heat transfer element permits efficient andrapid reaction cycles and can be used for thermal amplificationreactions (e.g., PCR) or isothermal reactions (e.g., rolling circleamplification, whole genome amplification, NASBA, or strand displacementamplification). In certain embodiments, the emulsion is flowed directlyinto the imaging region following continuous-flow amplification.

Single-molecule immunoassays and enzymatic assays may also be performedin droplets (see, e.g., Sakakihara et al., “A single-molecule enzymaticassay in a directly accessible femtoliter droplet array,” Lab on a Chip10:3355-3362 (2010); Sista et al., “Heterogeneous Immunoassays UsingMagnetic Beads On a Digital Microfluidic Platform,” Lab Chip.8(12):2188-2196 (2008)).

Working Examples

In a first working example of an exemplary embodiment according to thepresent disclosure, droplets were generated using an emulsificationdevice with a single multi-step nozzle having the following dimensions:CH=20 um, CW=60 um, SH1/CH=1.5 SH2/CH=1.75. The surfaces of themulti-step channel were coated with hydrophobicperfluorodecyltrichlorosilane (FDTS). In this example, a chemicallyinert oil (Fluorinert® FC-40) mixed with surfactant (to stabilize thedroplets; PFPE-PEG-PFPE) was placed in the device. A 2 μM solution ofoligonucleotides coupled to AP559 fluorescent dye in water was directedinto the inlet portion of the multi-step channel at a flow rate of 1-100nL/s. Droplets with a diameter of approximately 120 microns were formed,at a generation rate of 1 to 30 droplets per second and an averagedispersion percentage of approximately 3.8 percent.

In a second working example of an exemplary embodiment according to thepresent disclosure, an emulsification device with 99 nozzles was used toproduce droplets at a generation rate of approximately 20,000 dropletsper minute. In this example, the average diameter of the droplets wasapproximately 122 microns, and the dispersion rate was approximately 9percent. It is believed that the dispersion rate in this example washigher than expected due to a single, defective nozzle that producedinconsistent droplets. These nozzles were the same geometry used on thesingle nozzle part, CH=20 um, CW=60 um, SH1/CH=1.5, SH2/CH=1.75, dropletproduction rate of 1-30 droplets per nozzle per second. The continuousphase fluid was a solution of surfactant (PFPE-PEG-PFPE) in FC-40 andthe dispersed phase was a 2 uM solution of oligonucleotides coupled toAP559 fluorescent dye in water.

The above specification and examples provide a complete description ofthe structure and use of an exemplary embodiment. Although certainembodiments have been described above with a certain degree ofparticularity, or with reference to one or more individual embodiments,those skilled in the art could make numerous alterations to thedisclosed embodiments without departing from the scope of thisinvention. As such, the illustrative embodiment of the present devicesis not intended to be limited to the particular forms disclosed. Rather,they include all modifications and alternatives falling within the scopeof the claims, and embodiments other than the one shown may include someor all of the features of the depicted embodiment. Further, whereappropriate, aspects of any of the examples described above may becombined with aspects of any of the other examples described to formfurther examples having comparable or different properties andaddressing the same or different problems. Similarly, it will beunderstood that the benefits and advantages described above may relateto one embodiment or may relate to several embodiments.

The claims are not to be interpreted as including means-plus- orstep-plus-function limitations, unless such a limitation is explicitlyrecited in a given claim using the phrase(s) “means for” or “step for,”respectively.

REFERENCES

The following references are incorporated herein by reference:

-   Sugiura, “Interfacial Tension Driven Monodispersed Droplet Formation    from Microfabricated Channel Array”, Langmuir 2001, 17, 5562-5566.-   Dangla, “Droplet microfluidics driven by gradients of confinement”,    PNAS; Jan. 15, 2013; vol. 110, no. 3, 853-858.-   U.S. Pat. No. 5,736,330-   U.S. Pat. No. 5,981,180-   U.S. Pat. No. 6,057,107-   U.S. Pat. No. 6,268,222-   U.S. Pat. No. 6,449,562-   U.S. Pat. No. 6,514,295-   U.S. Pat. No. 6,524,793-   U.S. Pat. No. 6,528,165-   U.S. Pat. No. 7,842,457-   U.S. Pat. No. 7,927,797-   U.S. Pat. No. 8,048,627-   U.S. Pat. No. 8,296,088-   U.S. Patent Pub. 2013/0078164

1. An emulsification device comprising: a channel having an inletportion having a channel height CH and a width CW, wherein the ratio ofCW/CH is greater than 0.2 and less than 5.0; a first step in fluidcommunication with the inlet portion, wherein: the first step has atread length T1 and a step height SH1; SH1 is greater than CH by a riserheight R1; and the ratio of SH1/CH is greater than 1.0 and less than5.0; a second step in fluid communication with the first step, wherein:the second step has a tread length T2 and a step height SH2; SH2 isgreater than SH1 by a riser height R2; and the ratio of SH2/CH isgreater than 1.0 and less than 5.0; a third step in fluid communicationwith the second step, wherein: the third step has a step height SH3; SH3is greater than SH2 by a riser height R3; and R3 is greater than zero.2. (canceled)
 3. The emulsification device of claim 1 wherein the ratioof SH1/CH is greater than 1.0 and less than 2.0.
 4. (canceled)
 5. Theemulsification device of claim 1 wherein R3 is greater than 50 microns.6. (canceled)
 7. The emulsification device of claim 1 wherein the ratioof T1/CH is between 3.0 and 4.0.
 8. The emulsification device of claim 1wherein the ratio of T2/CH is between 2.0 and 4.0.
 9. The emulsificationdevice of claim 1 wherein CH is between 10 microns and 50 microns. 10.(canceled)
 11. The emulsification device of claim 1 further comprising:a plurality of inlet portions, wherein each inlet portion in the firstplurality of inlet portions has a height CH and a width CW, and whereinthe ratio of CW/CH is greater than 0.2 and less than 5.0.
 12. Theemulsification device of claim 11 wherein the plurality of inletportions comprises between 10 and 100 inlet portions.
 13. A method offorming an emulsion, the method comprising: obtaining an emulsificationdevice according to claim 1, wherein the first step, the second step andthe third step contain a first fluid that is substantially static; andintroducing a second fluid into the inlet portion and through the firststep, the second step and the third step, wherein: a partial droplet ofthe second fluid forms in the first step; a complete droplet of thesecond fluid forms in the second step; and the complete droplet of thesecond fluid is directed from the second step to the third step. 14.-16.(canceled)
 17. The method of claim 13 wherein the second fluid containsan analyte of interest and an assay reagent. 18.-20. (canceled)
 21. Themethod of claim 13 wherein the first fluid is a hydrophobic liquid andthe second fluid is a hydrophilic liquid.
 22. The method of claim 13wherein the first fluid is a hydrophilic liquid and the second fluid isa hydrophobic liquid.
 23. The method of claim 13 wherein the either thefirst fluid or the second fluid comprises an emulsifying agent.
 24. Themethod of claim 23 wherein the emulsifying agent comprises a non-ionicsurfactant or a blocking protein.
 25. (canceled)
 26. The method of claim13 wherein a complete droplet of the second fluid forms in the secondstep at a rate of between 1 and 30 complete droplets per second. 27.(canceled)
 28. The method of claim 13 wherein the complete droplet ofsecond fluid has an average diameter between 40 and 300 microns. 29.(canceled)
 30. The method of claim 13 wherein the emulsion formedbetween the first fluid and the second fluid has a monodispersitybetween two and ten percent. 31.-35. (canceled)
 36. A method of formingan emulsion, the method comprising: obtaining an emulsification deviceaccording to claim 11, wherein the plurality of first steps, theplurality of second steps and the plurality of third steps contain afirst fluid that is substantially static; and introducing a second fluidinto the plurality of inlet portions and through the plurality of firststeps, the plurality of second steps and the third step, wherein: apartial droplet of the second fluid forms in each of the plurality offirst steps; a complete droplet of the second fluid forms during thetransition between the plurality of first steps and the second steps ineach of the plurality of second steps; and the complete droplet of thesecond fluid is directed from the plurality of second steps to the thirdstep.
 37. The method of claim 36, wherein at least 10,000 completedroplets are directed from the plurality of second steps to the thirdstep per minute.
 38. The method of claim 37, wherein the droplets havean average dispersion of less than 10 percent.
 39. (canceled)
 40. Themethod of claim 36, wherein the average droplet diameter of droplets inthe third step is between 40 to 300 microns. 41.-63. (canceled)
 64. Theemulsification device of claim 11 further comprising: a plurality offirst steps, wherein each first step in the plurality of first steps is:in fluid communication with each of the plurality of inlet portions; andhas a length T1 and a height SH1, wherein SH1 is greater than CH by ariser height R1, and wherein the ratio of SH1/CH is greater than 1.0 andless than 5.0.
 65. The emulsification device of claim 64 furthercomprising: a plurality of second steps, wherein each second step in theplurality of second steps is: in fluid communication with a first stepin the plurality of first steps; in fluid communication with the thirdstep; and has a length T2 and a height SH2, wherein SH2 is greater thanSH1 by a riser height R2, and wherein the ratio of SH2/CH is greaterthan 1.0 and less than 5.0.
 66. The emulsification device of claim 11further comprising: a single continuous first step, wherein the singlecontinuous first step is: in fluid communication with each of theplurality of inlet portions; and has a length T1 and a height SH1,wherein SH1 is greater than CH by a riser height R1, and wherein theratio of SH1/CH is greater than 1.0 and less than 5.0.
 67. Theemulsification device of claim 66 further comprising: a singlecontinuous second step, wherein the single continuous second step is: influid communication with the single continuous first step; in fluidcommunication with the third step; and has a length T2 and a height SH2,wherein SH2 is greater than SH1 by a riser height R2, and wherein theratio of SH2/CH is greater than 1.0 and less than 5.0.